Squamous cancers of the cervix (SCCx) are caused by persistent infection with oncogenic strains of human papillomavirus (HPV). HPV16 is the strain associated with over 60% of malignant disease. The HPV E6 and E7 proteins are functionally required to maintain the transformed state, are consistently expressed in SCCx and in its precursor lesion, high grade cervical dysplasia (CIN2/3), and represent foreign antigens to the host. Therefore, they present potentially compelling immunotherapeutic targets. In collaboration with NCI RAID, we have made an HPV16E7-targeted therapeutic DNA vaccine. Our collaboration with Transgene, SA, will provide GMP-grade vaccine which consists of Modified Vaccinia Ankara (MVA), housing E6, E7, and IL2 (MVA-E6E7-IL2). Both of these vaccines have been tested singly in clinical trials in this patient population. We propose to evaluate heterologous DNA prime-MVA boost vaccination, with and without a topical Toll-like receptor (TLR) agonist applied at the lesion site, in otherwise healthy women with high grade cervical dysplasia associated with HPV16. This patient cohort is likely to be informative, as the rate of spontaneous regression in the study treatment window, 15 weeks, is expected to be 25% in this cohort. DNA-MVA prime-boost vaccination has been shown to be safe and immunogenic in clinical trials testing other antigenic targets, in healthy volunteers. We will test the hypothesis that DNA-MVA prime-boost vaccination is immunogenic and safe, and that locally applied TLR agonist can enhance cervical immune responses, allowing them to overcome immunologic inhibitory mechanisms present in the mucosal microenvironment of dysplastic lesions. The proposed analysis is novel because we will be able to study whether the generation of local inflammation is important in "unmasking" a chronic viral infection, and to study local, compartmentalized measures of effector and inhibitory immune responses. The study design targets a unique clinical resource and ideally suited patient population in which to demonstrate proof of principle. We have a demonstrated record in clinical trial design and execution in this patient population, which is disproportionately comprised of minority women who do not access the medical care system effectively. [unreadable] [unreadable] [unreadable]